Project Summary Development of an effective HIV-1 vaccine remains an unmet goal. Success in this endeavor will require a better understanding of how HIV-1 engages the adaptive immune system, including CD4+ T cells (TCD4+) that are instrumental in the development of effective humoral and cellular immunity. TCD4+ are activated by antigen-derived peptides complexed with Major Histocompatibility Complex class II molecules (MHC-II). According to convention, these peptides are derived from extracellular antigen that is degraded and loaded onto nascent MHC- II in the endosomal compartment of an antigen presenting cell (APC). However, our work with influenza and ectromelia viruses indicates that peptide generation is far more complex, with several fundamentally distinct antigen processing modes in play. Division of labor between these modes is governed by parameters such as physical properties of the virion and replication strategy, which is unique to each virus. How this division plays out for HIV-1 is unknown, since surprisingly little work has been done in this area despite the potential for major impact on rational vaccine design. Here we propose to elucidate the MHC-II processing landscape for HIV-1, and initiate investigation of the underlying processing machinery. This R21 funding mechanism will serve as a springboard for continued mechanistic studies in one direction, and the application of emerging concepts to rational vaccine design in the other.